Prevalence of plasmid-encoded carbapenemases in multi-drug resistant Escherichia coli from patients with urinary tract infection in northern Iran

Objective(s): Resistance to carbapenems as the last line for controlling resistant bacteria is increasing due to production of carbapenemase. The aim of this study was to detect the plasmid-encoded carbapenemases using phenotypic methods and multiplex PCR among the multi-drug resistant (MDR) isolates from patients with urinary tract infection (UTI) in northern Iran. Materials and Methods: Antimicrobial susceptibility testing and extended spectrum β-lactamase (ESBL) production test were performed for 91 MDR Escherichia coli strains by disc diffusion and double disk synergy tests (DDST), respectively. Carbapenemases production was confirmed using Hodge test, EDTA double disk synergy test (EDST) and combined disk test (CDT). The isolates were subjected to PCR targeting blaIMP, blaVIM, blaKPC and blaOXA-48 β-Lactamase genes. Results: Resistance of isolates to 1st, 2nd, 3rd, and 4th generations of cephalosporins, carbapenems, and penicillins were 73%, 84.5%, 62%, 37.5%, 17.5%, and 76%, respectively. Based on CDT and Hodge test, 1 (3%) and based on EDST, 2 (6%) of 33 ESBL producers synthesize a type of carbapenemase. The frequency of blaIMP, blaVIM, blaKPC, and blaOXA-48 genes was 8.7%, 9.8%, 2.1%, and 15.3%, respectively. Existence of blaIMP conferred more resistance to cephalotin, fosfomycin, and piperacillin (P≤0.01) and carrying blaVIM caused more resistance to cephalotin, cefepime, and ceftazidime (P≤0.01). The presence of blaKPC conferred more resistance to cephalotin and presence of blaOXA-48 caused more resistance to chloramphenicol and piperacillin (P≤0.05). Conclusion: Identification and controlling of this nearly low frequent ESBL and carbapenemase producing strains are important due to the presence of plasmid genes encoding carbapenemase.


Introduction
Over the last decade, the emergence of resistance to carbapenems, has become a major public health crisis worldwide especially in developing countries, due to their rapid spread and the lack of development of new antimicrobial agents (1)(2)(3). Resistance to carbapenems was reported in 86% of Gram-negative bacterial strains in Iran in 2010 (4).
Throughout 2006-2018, incremental trend of resistance to carbapenems was evident in Iran (5). The least rate of resistance was reported in year 2010 at Milad Hospital (6). During years 2012-2015, a study evaluated the trend of antibiotic resistance in Acinetobacter baumannii. Based on their study results, resistance rate in A. baumannii increased from 83% in year 2012 to 96% in year 2015. Also 100% of A. baumannii isolates during these years were resistant to carbapenem (7). No specific trend was followed by the other microorganisms' resistance patterns. Most of the carbapenem-resistant strains were isolated from burn patients, and many studies which were conducted in this group were from Motahari Hospital, Tehran, Iran (5).
Since 1993, wide varieties of carbapenemases have been recognized that belong to three molecular classes: the Ambler class A, B, and D β-lactamases (3).
In this investigation, four carbapenemases including IMP, VIM, KPC, and OXA-48 were studied. KPC stands for Klebsiella pneumoniae carbapenemase and is a class A β-lactamase that has the ability to hydrolyze penicillins, cephalosporins, and carbapenems. KPC was initially reported from a K. pneumoniae strain isolated in North Carolina in 1996 (8).
The IMP-type enzymes, initially reported in 1991 in a Serratia marcescens clinical isolate from Japan (2), have now been reported all over the world in Enterobacteriaceae, Pseudomonas aeruginosa, and A. baumannii (2). The most commonly found class B carbapenemases are of the VIM type, which has been identified in all continents (2). The death rates associated with MBL producers are high (18% to 67%) (10,11).
OXA stands for oxacillinase and is a diverse group of β-lactamases classified to class D. Some of OXA β-lactamases additionally have the capability to hydrolyze carbapenems. OXA-48 was first found in a K. pneumoniae strain isolated in Turkey in 2001 (12). Its production mediates resistance to penicillins and carbapenems (especially imipenem), but not to cephalosporins. In Iran, OXA-48 was first reported in 2017 in the Escherichia coli isolates (13).
Among the uropathogenic bacteria, E. coli is predominant in both community and nosocomial urinary tract infection (UTI) (14)(15)(16). These resistance patterns have shown large inter-regional variability. Understanding the spectrum and resistance patterns may help guide effective empirical antibiotic therapies and decrease treatment failure and costs.
Contact precautions and outbreak detections require reliable detection of carbapenemases. However, detection of carbapenemase in Enterobacteriaceae is challenging, because carbapenemase-producing K. pneumoniae with low carbapenem MICs have been described in the CLSI or EUCAST-susceptible range. Also, a difference between porin loss coupled with an ESBL or AmpC β-lactamase or carbapenemase is not feasible in carbapenem-resistant isolates alone on the basis of the antibiogram (17). Phenotypic tests such as the modified Hodge test are helpful to detect carbapenemases but have low sensitivity for NDM and low specificity (18). Phenotypic tests based on synergy with EDTA are available for detection of MBL but can produce false-positive outcomes with certain strains and cannot distinguish between kinds of MBL (19). Class A carbapenemases such as KPC can be identified through synergy with boronic acid, but if AmpC β-lactamases are coproduced, false-positive synergy test findings occur (20). Thus, confirmation using molecular analysis is essential.
Due to limited information on carbapenemase in Iran (21), identifying the resistant strains is a major challenge for diagnostic laboratories. The carbapenemases that were surveyed in this study were encoded by plasmids and due to their transfer to other isolates, the purpose of this study was to identify types of carbapenemases using phenotypic methods and to determine the frequency of plasmid genes encoding carbapenemases (IMP, VIM, KPC, and OXA-48) among the MDR isolates causing UTI in northern Iran.

Bacterial isolates
Urine samples of the patients (138 samples including 31 male and 107 female specimens with mean age of 43 for male and 41 for female) were collected from appropriate patients in early morning mid-stream using sterile, wide mouthed glass bottles with screw cap tops between May and July 2017. Samples were maintained in an icebox until laboratory analysis. Sample collection and its analysis were no more than one hour apart. The usual bacteriological methods were applied for cultivation, isolation and identification of the strains. Urine samples were cultured on Nutrient Agar, Blood Agar, Eosin Methylene Blue Agar (EMB), and MacConkey agar plates and incubated at 37 °C for 18-24 hr. Urine culture was considered positive, if it contained ≥10 5 cfu/ml. E. coli from positive urine cultures identified by their characteristic appearance on the media, Gram staining reaction, by the pattern of biochemical tests such as catalase, oxidase, ONPG, IMViC tests, lactose fermentation, H 2 S and CO 2 production, urea hydrolysis, and lysine decarboxylase (22). The isolates were stored at -70 °C in a Tryptic Soy Broth containing 15% glycerol until processing.

Antibacterial susceptibility testing
To identify the susceptibility of the isolates to antibiotics, the disc diffusion test was used according to Clinical and Laboratory Standards Institute (CLSI) (23) guidelines; the following antibiotics were utilized: ampicillin (AMP) ( . E. coli ATCC 25922 and ATCC 35218 were used as the standard strains to control the quality of the applied antimicrobial agents. MDR is defined as resistance to three or more antibiotics.

Detection of ESBL
In order to identify ESBL, double disk synergy test (DDST), which depends on comparing the inhibition zone given by CAZ (30 µg) and CAZ-plus-clavulanate (30 µg/10 µg) was used. A difference of ≥5 mm between the zone of CAZ-plus-clavulanate and CAZ alone was taken to indicate ESBLs production as advocated by CLSI (23).

Hodge test
Briefly, a 0.5 McFarland bacterial suspension of E. coli ATCC 25922 as control or susceptible strain was inoculated on the surface of a Mueller-Hinton agar (MHA) as a lawn culture. After brief drying, a 10 µg imipenem disk was placed at the center, and the test isolate was streaked from the center to the periphery of the plate and the plate was incubated overnight. Isolates which produced a cloverleaf-shaped inhibition zone were recognized as producers of carbapenemase (24).

Imipenem-EDTA combined disk test (CDT)
As recommended by CLSI, the control strain was cultured as a lawn on the MHA plate along with test isolates (turbidity of 0.5 McFarland). Then, two 10-μg meropenem discs were located on the lawn culture with 20 mm distance from center to center of the discs. In one of the meropenem disks, a 10 μl 0.5 M EDTA was added and incubated overnight. Isolates indicating a rise of ≥7 mm in the meropenem+EDTA disc's inhibition zone size compared to the meropenem disc alone were known MBL producers (24).

EDTA-disk synergy test (EDST)
An overnight broth culture of the test isolate was suspended to the turbidity of a 0.5 McFarland and used to swab inoculate a MHA. A 10-μg meropenem disc and a blank disk (Whatmann filter paper no. 2, 6 mm in diameter) were located 10 mm apart from edge to edge, 10 μl EDTA solution 0.5 M was then used as the blank disc. The plates were incubated at 37 °C overnight and an expanded inhibition zone was interpreted as positive EDS (24).

Multiplex PCR technique
DNA extraction was performed with suspending one colony in 100 µl of distilled water (95 °C for 10 min) followed by centrifugation of the cell suspension.
The DNA concentration and purity were determined by spectrophotometric measurement of absorbance at 260 and 280 nm by a UV spectrophotometer. All DNA samples were dissolved in water and stored at -20 °C Table 1.
Amplification was carried out with the following thermal cycling conditions: 10 min at 94 °C and 30 cycles of amplification consisting of 94 °C for 40 sec, 60 °C for 40 sec, and at 72 °C for 1 min, with 7 min at 72 °C for the final extension. For the multiplex PCR analysis, the annealing temperature was at 55 °C for amplification of bla VIM , bla IMP , and bla KPC genes, and 57 °C for amplification of bla OXA-48 gene. The PCR products were subjected to electrophoresis on a 2% agarose gel and observed under UV after staining with ethidium bromide (25).

Statistical analysis
The data were statistically analyzed using One-way analysis of variance (ANOVA) and differences among the means were determined at P≤0.01 using Duncan's multiple range tests (by SAS, 9.1).

Isolates and their resistance patterns
From 138

ESBL and carbapenemase detection
Of total 91 E. coli isolates, the synthesis of ESBL was detected in 33 isolates. In this study, phenotypic and genotypic tests were carried out for detection of different types of carbapenemases including IMP, VIM, KPC, and OXA-48. Based on the Hodge test, 3 (3%) of the 33 ESBL-producing E. coli isolates produced imipenemase. Using CDT and EDST methods, 1 (3%) and 2 (6%) of the 33 ESBL-producing E. coli isolates produced MBL (IMP, VIM, or both and/or other MBLs), respectively. Therefore, these two methods have somewhat different Table 1. Group-specific primers used for multiplex PCR (23) bY=T or C; R= A or G; S= G or C; D= A or G or T  performances in MBL detection. It was interesting that by applying the phenotypic methods in this study, it was proven that none of the non-ESBL-producing E. coli isolates were able to produce different types of carbapenemases.

Discussion
Resistance to carbapenems is due to carbapenemase and other resistance mechanisms, such as ESBLs, efflux pumps, and/or porin mutations (10). The current emergence of carbapenemase-producing bacteria especially Enterobacteriaceae is of concern because it is often associated with the occurrence of multidrug-resistant isolates, where there are very few drug options available for them, if any (10). Therefore, detection and initial identification of carbapenemaseproducing bacteria are important. In some cases, due to the low sensitivity or specificity of phenotypic methods, molecular approaches may also be used (26). Reliable identification of carbapenemases is essential for the implementation of contact precautions and the detection of the outbreak.
In the current study, from the 112 urine specimens from UTI patients infected with bacteria, 91 were positive for E. coli. Carbapenems and pipiracillin were the most effective antibiotics and all cephalosporins other than 4 th cephalosporin affected more than 50% of isolates. Compared to the ESBL-producing isolates, resistance of non-ESBL-producing isolates was higher than different antibiotics. This is due to another mechanism other than ESBL in resistance to antibiotics. Generally, the frequency of ESBL-producing isolates and the types of carbapenemaese genes among them were low and also in the detection of carbapenemases, there was no correlation between the results of phenotypic and molecular analyses. Due to the similarity of the results of the Hodge and CDT tests, it seems that in the Hodge test, the addition of EDTA did not have any effect on the improvement of the test. Also, the EDST test is comparison with CDT could detect more MBLs and therefore, EDST probably is more reliable. The results of current study in the detection of cabapenemases types in ESBL-producing E. coli showed that multiplex PCR is both more sensitive and also more reliable than phenotypic methods due to detection of different carbapenemases and more positive samples.
Given that the resistance of the isolates to imipenem and moropenem was 29% and 6%, respectively, the multiplex analysis identified fewer resistance genes to carbapenems (frequency of OXA-48, KPC, IMP, and VIM genes was 7% (10/33), 1% (2/33), 4% (6/33), and 4% (6/33), respectively), which was similar to that of Gheitani and Fazeli (21). The possible reason for this may be the presence of other types of resistance genes to carbapenems that have not been investigated in the current study or other mechanisms of resistance to carbapenem other than carbapenemase production.
In a study to detect MBL among 100 A. baumannii strains, 30 (30%) of the strains were positive for the bla VIM gene, but the bla IMP gene was not detected in any of the strains (46).

Conclusion
Although the prevalence of the ESBL-producing strains and the simultaneous presence of several carbapenemase genes in the studied population were nearly low, these low prevalent strains and genes are responsible for resistance to some antibiotics. Thus, identification and controlling of these strains is important due to the presence of plasmid genes encoding carbapenemases and their easy transferability to other clinical isolates.